利用卒中iPSC模型研究基因组杂合性缺失对X染色体倾斜性失活的调控

X-chromosome inactivation (XCI) is one of the most important epigenetic modifications, and results in random transcriptional inactivation of one of the two X chromosomes present in normal, female mammalian cells. This process allows mammals to achieve dosage equivalence of most X-linked genes between females, who normally have two X chromosomes, and males,who normally have one X chromosome. However, XCI might not always be random and that abnormal skewed status, which termed SXCI usually lead to some certain diseases, such as Alzheimer's disease and cancer. Studies of mechanism of SXCI occurrence is still limited but of urgency. It has been demonstrated that genome micro-abnormalities such as copy number variation (CNV),loss of heterozygosity (LOH) and single nucleotide polymorphisms (SNPs) were the main causes of human disease. According to our previous study, it's found that a high degree of correlation between the incidence of stroke and the SXCI status, and there were some common genome abberations of LOH in the X chromosome among the stroke, where are enriched OMIM genes of nervous system. There are, however, no published data that directly assess the status of XCI are related to stroke. We speculated that LOH in the genome，especially in X chromosome, might cause skewed X chromosome inactivation which lead to a disorder gene expression of the nervous system and resulted in corresponding diseases. To examine this hypothesis, a good model for gene function study should be firstly developed. Induced pluripotent stem cell (iPSC) derived from somatic cells of patients represent a powerful tool for biomedical resaerch of gene function studies and may have a wide range of applications in cell and gene therapy based on its unique properties including self-differentiation, self-renewal and multi-directional differentiation. We intend to take the stroke patients as the object, establish the corresponding iPSC by the Kyoto method and . To claim a novel occurrence mechanism, beside the known site mutations, of the skewed X chromosome inactivation, the genome and the transcriptome were analysed combining the whole genome chips and RNAseq etc. The expression of the nervous system OMIM genes as well as newly identified candidates, which located in a highly relevant LOH, would be investigated to clarify the association between SXCI and the nervous system diseases. RNAi of lncRNA, including Xist, Ftx, Jpx and Tsix, would be performed to check whether they influenced the expression and regulation of the nervous OMIM gene.

X染色体失活是人类表观遗传研究的重要内容，异常的X染色体倾斜性失活(SXCI)通常会引发老年痴呆、癌症等疾病。然而关于SXCI发生机制的研究仍相当有限。基因组微失衡是人类疾病主要成因，其形式包括CNV、SNP及LOH（杂合性缺失）。课题组前期研究发现，卒中发病与SXCI有高度相关性，且在X染色体结构上存在共同的LOH，而该LOH区段富集了一系列神经系统致病基因。我们推测，LOH会引起SXCI并导致部分神经系统致病基因表达异常，从而诱发疾病。 多能性干细胞（iPSC）是研究基因功能的优良模型。本课题拟建立卒中iPSC疾病模型，结合芯片及RNAseq技术，揭示LOH是否为点突变以外新的SXCI发生机制，对高度相关的LOH区域所含神经系统致病基因的表达进行分析，明确SXCI与神经系统疾病发生的关联性及指向性，鉴定新分子靶标及lncRNA对该级联反应的调控作用，探索卒中疾病的表观遗传调控机制。

缺血性脑卒中严重降低了人类生活质量，给社会带来恶劣的负面影响。据预测，截至2050年，女性脑卒中发病率将比男性高出30%。通过对女性缺血性卒中的危险因素的研究及从而达到预防目的是有意义的。X染色体失活是表观遗传学的重要组成部分，并与多种人类疾病相关，但尚无关于其与脑卒中发病的相关性研究。本项目共采集了100名缺血性脑卒中女性患者与100名年龄无统计学差异的健康对照人群的外周血，对其中AR为杂合子的个体进行X染色体失活模式鉴定；并对具有家族史的倾斜性失活病人进行染色体芯片及甲基化芯片分析以明确其基因组失衡情况及DNA甲基化水平；结合胚胎干细胞的结果推导出与X染色体倾斜性失活相关的LOH区域，并分析JPX等相关基因的表达和调控因素；建立了女性患者iPS细胞6株并对它们进行诱导分化。结果显示，在缺血性脑卒中86例AR基因为杂合子的患者中，SXCI患者有30例，占33.7%，而对照组中的数量为10例，比例为11.6%，X染色体倾斜性失活的比例在卒中组显著升高（P=0.021）。患者在基因组上并没有出现聚类的CNV，也未检测到已知导致X染色体倾斜性失活的XIST基因-43G-A突变，但均在Xq11.2-Xq22.1区域检测到中性的LOH，涉及XIST、TISX、JPX和FTX四个lncRNA。甲基化芯片结果显示，发现三名随机失活的脑卒中患者的整体甲基化水平的坐标分布更为接近，X染色体随机失活及倾斜性失活的脑卒中病人在DNA甲基化上的表观调控仍有叫显著的差异（DMR>0.1，p<0.05）。DMR比例为2.44%，其中位于X染色体上的DMR有191个，包括了JPX和TSIX的调控区。qPCR表明，XIST在倾斜性失活的细胞株中表达明显增加，而JPX在则倾斜性失活的细胞株表达降低，而另外2个lncRNA未发现显著的表达差异。所建立的Is-iPS细胞株均保持正常核型，表达多能性基因并能在体内形成畸胎瘤，而且表现出倾斜性失活的特质。对Is-iPS进行诱导造血分化，其效率为15.9%，与对照无差别。本研究首次证实了X染色体倾斜性失活与脑卒中女性患者发病的相关性。这将对今后女性Is人群的预警、早期发现以及一级预防有重要的理论价值。所鉴定的Xq11.2-Xq22.1区域的LOH补充了X染色体倾斜性失活发生的遗传性高危因素。所建立的Is-iPS可作为进一步研究内源性凝血系统功能的良好模型。